NIR-Absorbing Dye Functionalized Supramolecular Vesicles for

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NIR-Absorbing Dye Functionalized Supramolecular Vesicles for Chemo-Photothermal Synergistic Therapy Qi Wang, Peng Zhang, Jingzeng Xu, Bing Xia, Lu Tian, Jiaqi Chen, Jie Li, Feng Lu, Qingming Shen, Xiaomei Lu, Wei Huang, and Quli Fan ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00014 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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ACS Applied Bio Materials

NIR-Absorbing Dye Functionalized Supramolecular Vesicles for Chemo-Photothermal Synergistic Therapy Qi Wang,† Peng Zhang,† Jingzeng Xu,† Bing Xia,† Lu Tian,† Jiaqi Chen,‡ Jie Li,† Feng Lu,† Qingming Shen,† Xiaomei Lu,‡ Wei Huang†,‡ and Quli Fan*,† †

Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory

for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China ‡

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China KEYWORDS: supramolecular vesicles, pillar[5]arene, chemotherapy, photothermal therapy, synergistic therapy ABSTRACT: Developing multifunctional supramolecular nanoplatforms for effective tumor therapy is a challenging task. In this contribution, a NIR-absorbing perylene diimide dye G has been prepared, which not only acted as a photothermal agent but also served as a guest. Multifunctional supramolecular vesicles were efficiently constructed by the recognition of dye G and water-soluble pillar[5]arene, which showed high-loading capacity for hydrophobic chemotherapy drug doxorubicin. What's more, the resulting drug-loaded vesicles had high stability in simulated normal physiological condition but showed rapid drug release in the acidic microenvironment. Additionally, the drug-loaded vesicles exhibited more remarkable antitumor efficacy than chemotherapy or photothermal treatment alone through chemo-photothermal synergistic therapy. Furthermore, such supramolecular drug nanocarriers could efficiently get inside the tumor cells mainly via endocytosis to obtain excellent accumulation of drug in tumor

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sites. Overall, this study offers an innovative tactic to fabricate smart nanocarriers for synergistic therapy of cancer.

INTRODUCTION Although traditional chemotherapy has still been the primary choice for clinical treatment of various malignant tumors, its therapeutic efficacy has markedly hindered by many drawbacks, such as non-specificity, low bioavailability, severe side effects and drug resistance.1-3 In order to mitigate the inadequacy of chemotherapy, synergistic therapy strategy can integrate multiple therapeutic modalities, providing a simple and efficient modality for cancer treatment.4-6 In recent years, photothermal therapy (PTT), which relies on cytotoxic heat generated by irradiation of photothermal agents with tissuepenetrating near-infrared (NIR) light to ablate tumor cells, has potential advantage in the enhancement of chemotherapy efficacy.7-12 In particular, perylene diimide (PDI)-based NIR-absorbing dyes have become a promising photothermal agent in clinical practice because of their superb biocompatibility, easy modiﬁcation, excellent photothermal conversion ability and extremely low cost.13 For instance, our group developed novel PDI-based polymer nanoparticles for photothermal therapy, which shown excellent antitumor efficacy.14 Up to now, various nanocarriers have been developed to synergistically combine phototherapy and chemotherapy.15-21 However, the integration of PDI-based NIR-absorbing dye with antitumor chemotherapy drug via supramolecular vesicles for multimodal synergistic cancer therapy has not been well studied. Supramolecular vesicles have exhibited more and more advantages as smart drug delivery systems (DDSs) owing to their unique abilities to load and controllably deliver drugs at pathological sites with specific microenvironmental changes.22-25 Pillararenes,26,27 an important kind of supramolecular hosts with unique symmetrical pillar architecture, excellent host-guest binding abilities and stimuli-responsive properties, have been adopted extensively for the construction of functional supramolecular materials,28-32 especially the supramolecular vesicles.33 However, most of these studies employed only single chemotherapy, which showed limited therapeutic efficacy.34,35 In order to achieve a better therapeutic outcome, herein, we designed a NIR-absorbing PDI dye G that not only acted as a photothermal agent but also served as a guest. Multifunctional


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supramolecular vesicles were efficiently fabricated by the recognition of dye G and watersoluble pillar[5]arene (WP5), which showed good loading capacity for hydrophobic chemotherapy drug doxorubicin (DOX) and effective drug release in low-pH tumor condition. Moreover, these supramolecular drug nanovehicles could efficiently get inside the tumor cells via endocytosis to achieve high accumulation of drug in tumor location and display synergistically chemo- and photothermal therapy, indicating a suitable nanoplatform for synergistic therapy of cancer (Scheme 1). Scheme 1. Cartoon Representation of the Multifunctional Supramolecular Vesicles for ChemoPhotothermal Synergistic Therapy

RESULTS AND DISCUSSION Host-guest Recognition Study. A well-designed guest G was prepared in a four-step reaction (Scheme S1), while WP5 was prepared according to previous method.36 To study the recognition between G and WP5 through 1H NMR spectroscopy, GM was prepared as a model guest because of the self-aggregation behavior of G. In Figure 1, after mixing WP5 and GM, the protons H1, H2, H3 and H4 of WP5 moved downfield slightly (∆H1 =


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0.15 ppm, ∆H2 = 0.09 ppm, ∆H3 = 0.03 ppm and ∆H4 = 0.08 ppm). Whereas, the protons Ha, Hb, Hc and Hd of GM shifted upfield remarkably (∆Ha = −0.21 ppm, ∆Hb = −0.55 ppm, ∆Hc = −0.48 ppm and ∆Hd = −0.20 ppm), confirming that the GM was threaded through the hydrophobic cavity of WP5 to produce a threaded WP5⊃GM complex. The pHresponsiveness of WP5⊃GM was proved by 1H NMR spectroscopy (Figure S7). The carboxylate groups on both rims of WP5 were protonated after decreasing pH value of solution, making the host precipitate in water. The chemical shifts of protons on GM almost returned to their original uncomplexed positions, confirming the dissociation of the host-guest complex. However, after further adding NaCO3, the complexation between WP5 and GM was recovered, which could be discerned by the changes in chemical shifts of GM. Job plots based on UV-vis spectroscopy confirmed that WP5⊃GM exhibited 1:1 stoichiometry (Figure S8). The association constant for WP5⊃GM was measured about (1.15 ± 0.28) × 103 M–1 according to 1H NMR titration experiments (Figure S9-Figure S10).

Figure 1. 1H NMR spectra (298 K, 400 MHz, D2O): (a) GM; (b) 5.0 mM GM and 5.0 mM WP5; (c) WP5. Fabrication of Supramolecular Vesicles. After establishing WP5⊃GM recognition motif, a 2:1 bola-type supra-amphiphile could be obtained by forming a (WP5)2⊃G


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inclusion complex, and then the capability of such supra-amphiphile to fabricate supramolecular vesicles in aqueous solution was further studied. Because G itself is a bola-type amphiphilic molecule, the aggregation behavior of G was first studied. The solution of G showed a notable Tyndall effect when its concentration exceed 4.68 × 10−6 M (critical aggregation concentration (CAC), Figure S11a), demonstrating the formation of abundant nanoaggregates (Figure 2b). Then the morphology and size of such nanoaggregates were further studied. Solid spheres about 60 nm were discovered in TEM image (Figure 2a), which was close to the DLS result (about 63.5 nm, Figure 2b). After mixing WP5 and G, a distinct Tyndall effect of (WP5)2⊃G could also be observed (Figure 2e), which suggested the formation of nanoparticles. Moreover, [WP5]/[G]=1:5 was chosen as the best molar ratio for fabricating these nanoparticles (Figure S12). Under optimal molar ratio, the CAC for (WP5)2⊃G inclusion complex was deemed about 2.84 × 10−5 M (Figure S11b). TEM image of (WP5)2⊃G complex clearly showed the formation of vesicular structure aggregates of about 280 nm (Figure 2c), which was close to the DLS result (about 295.0 nm, Figure 2e). Additionally, the wall thickness of vesicles was deemed about ca. 5 nm (Figure 2d), fit nicely with the extended length of the (WP5)2⊃G complex estimated by Chem 3D (Figure S13), indicating that the vesicles had a monolayer wall as shown in Scheme 1. Moreover, ζ-potential assays suggested that the formed supramolecular vesicles at the best molar ratio had a large negative ζ-potential (-30.7 mV, Figure S14), indicating the obtained vesicles were relatively stable in aqueous solution. The long-term stability of supramolecular vesicles in PBS solution was also tested. With time, the hydrodynamic diameter of vesicles remained unchanged, suggesting excellent long-term stability of vesicles (Figure S15). Furthermore, no vesicles but solid nanoparticles about 60 nm were observed after changing pH to 6.0, indicating a good pH-responsiveness (Figure 2f).


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Figure 2. (a) TEM image, (b) DLS result and Tyndall effect of G in water; (c) TEM image of vesicles formed by WP5 and G; (d) enlarged image of c; (e) DLS result and Tyndall effect of vesicles formed by WP5 and G; (f) TEM image of (c) after further changing the solution pH to 6.0. Drug Encapsulation and Its Controllable Release. An outstanding nanoparticle for DDS should possess high stability in normal physiological condition but respond to pathological sites with internal or external stimuli for controlled release of drug. In view of the pH-responsiveness of the WP5, hydrophobic chemotherapy drug DOX was used to investigate the drug loading ability of the formed vesicles and their pH-responsive controlled release behaviour. In Figure 3a, after loading DOX to vesicles, the emission intensity of DOX-loaded vesicles around 520 nm to 660 nm became remarkably higher than unloaded vesicle solution after changing pH to 3.0 or adding Triton X-100 to realize totally discharge encapsulated DOX. Meanwhile, DLS (Figure 3b) and TEM (Figure 3c) results of DOX-loaded vesicles showed that their average diameter was larger than those of blank vesicles, further demonstrating that the drug was succeeded in loading into the supramolecular vesicles. Moreover, from fluorescence spectra, the drug encapsulation efficiency of the formed supramolecular vesicles was deemed about 27.2%, revealing that this system has a relatively large loading capacity for hydrophobic chemotherapy drugs. Next, the controlled DOX release form DOX-loaded vesicles in different acidic conditions were studied by fluorescence spectroscopy. In Figure 3d, the release percentage of DOX


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molecules was only about 18.2% in 24 h under simulated normal physiological condition (pH 7.4), demonstrating that the drug-loaded vesicles possess high stability in normal physiological condition. Nevertheless, under acidic environments at pH 6.2 and 4.4, rapid release of DOX could be observed, with the release percentages of 62.4% at pH 6.2 and 80.0% at pH 4.4 in the 24 h, respectively. The rapid DOX drug release behavior in acidic condition can be explicated by pH-induced supramolecular vesicles collapse due to the insoluble host production after protonates the carboxylate units on WP5, accompanying the rapid release of DOX. Compared with normal tissues, the tumor tissues show more acidic microenvironments, increased release of drugs induced by the acidic microenvironment is a very helpful feature for specific cancer treatment. Therefore, the rapid release behavior under acidic condition and good stability under normal physiological condition, reflecting this system suitable candidate for smart DDS.

Figure 3. (a) Fluorescence spectra of blank vesicles and drug-loaded vesicles at neutral condition (pH 7.4), acidic condition (pH 3.0), and dealt with Triton X-100; (b) DLS result and (c) TEM image of drug-loaded vesicles; (d) Controlled release behavior of drugloaded vesicles in different acidic conditions. In vitro NIR-Induced Photothermal Conversion Performance Studies. The UV-vis-NIR spectrum show that the supramolecular vesicles have good NIR absorption (Figure S16a),


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indicating they might convert NIR light into heat.37-41 The supramolecular vesicles exhibit an excellent molar absorption coefficient (8.34×103 M−1 cm−1) at 680 nm (Figure S17) and high photostability (Figure S18), demonstrating that the vesicles are good NIR-absorptive materials for PTT. To confirm the photothermal efficiency of the vesicles, the change of temperature during laser exposure in vitro was primarily evaluated using an infrared thermal imaging camera. The photothermal conversion performance of the vesicles with various concentrations under 730 nm NIR laser (1.5 W/cm2) irradiation was firstly investigated. As exhibited in Figure 4a, the supramolecular vesicles exhibited obvious concentration-dependent heating effect. The higher vesicle concentrations led to higher temperature increases. Upon light irradiation for 10 min, the water’s temperature merely increased by 3.3 °C, while the temperature of vesicles ([G] = 0.20 mM) quickly increased to 62.4 °C, which could efficaciously damage cancer cells. The photothermal conversion efficiency (η) of the vesicles was deemed about 44.5%, which is greater than that of many photothermal agents.42 The temperature increase curves of the vesicles after light irradiation for different times can be verified by the corresponding infrared thermal images (Figure 4c). Moreover, the rate of temperature elevation of the vesicles was proportional to the laser power density (Figure 4b), validating that the photothermal efficiency of the vesicles was also laser power density-dependent. Therefore, it would be convenient that the hyperthermia temperature can be effectively controlled by adjusting the concentration of vesicles or laser power density or both. By repeating the on-and-off cycle of the NIR laser irradiation, no appreciable change was observed, suggesting that the supramolecular vesicles can keep stable photothermal efficiency even after six cycles (Figure 4d). Similar behavior was also observed for G, indicating that G also has immense potential as photothermal antitumor agent (Figure S19).


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Figure 4. Photothermal profiles of supramolecular vesicles (a) with different concentrations after 730 nm laser irradiation (1.5 W/cm2) and (b) after 730 nm laser irradiation with different power density; (c) infrared thermal images and (d) photostability of supramolecular vesicles ([G] = 0.10 mM) under 730 nm laser irradiation (1.5 W/cm2). Cytocompatibility and Synergistic Cancer Therapy Assay. For potential biomedical and clinical application, an evaluation of the biocompatibility and synergistic therapy of as-prepared materials is an essential prerequisite. According to wang’s report, WP5 exhibited good biocompatibility in water,43 therefore the biocompatibilities of guest G, unloaded vesicles and drug-loaded vesicles toward MCF-7 cells were investigated by MTT assay, and the biocompatibility of DOX was also studied as a control. As exhibited in Figure S20, DOX shows similar cell cytotoxicity whether in the dark or upon irradiation. Moreover, no obvious cell cytotoxicity of G or unloaded vesicles was observed even at high concentration (100 µM, Figure 5a), suggesting excellent biocompatibility of this supramolecular drug nanocarrier. In contrast, a markedly higher cytotoxicity can be observed for both G and unloaded vesicles upon light irradiation (730 nm) because of the PTT effect. Moreover, the relative cell viability incubated with DOXloaded vesicles was significantly lower than that of unloaded vesicles, indicating that the encapsulated

DOX

could

be

successfully

released


under

an

acidic

tumor 9


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microenvironment (Figure 5b). More importantly, under 730 nm NIR laser irradiation, the DOX-loaded vesicles exhibited higher cytotoxicity than the chemotherapy or PTT treatment alone because of the synergistic cytotoxic effect of DOX and the photothermal effect of guest G. Furthermore, the morphology of living MCF-7 cancer cells disclosed conclusions similar to these mentioned above (Figure 6). All these results unambiguously demonstrate that the DOX-loaded vesicles are efficient and biocompatible supramolecular nanocarriers for synergistic cancer therapy.

Figure 5. In vitro cytotoxicities of (a) guest G and unloaded vesicles, and (b) drug-loaded vesicles against MCF-7 tumor cells in dark or in 730 nm laser irradiation (1.5 W/cm2), respectively. The error bars represent the standard deviation of three separate measurements (*p < 0.05, **p < 0.01).

Figure 6. Images of living MCF-7 cells in (a) control; (b) control with 730 nm NIR light; (c) G; (d) G with 730 nm NIR light; (e) unloaded vesicles; (f) unloaded vesicles with 730 nm NIR light; (g) DOX-loaded vesicles; (h) DOX-loaded vesicles with 730 nm NIR light after incubating for 24 h ([G] = 100 µM, 730 nm, 1.5 W/cm2). Scale bars = 20 µm.


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Cellular Uptake and Intracellular Localization. Confocal laser scanning microscopy (CLSM) was further applied to study the cellular internalization of drug-loaded vesicles toward MCF-7 cancer cells. Because drug DOX has inherent red fluorescence, the fluorescence intensity of cells that treated with drug-loaded vesicles can be clearly observed to monitor the uptake amount of DOX. Meanwhile, Hochest (shown in blue) and Lyso-Tracker Red (shown in green) were chosen to stain the nucleus and lysosomes, respectively, to locate these vesicles. Initially, after incubation for 1 h, weak red fluorescence of drug in MCF-7 tumor cells can be observed (Figure 7), suggesting that drug-loaded vesicles have already got inside the MCF-7 tumor cells. Whereas the intensity of drug in MCF-7 tumor cells increased significantly after 5h incubation, indicating that the uptake amount increased with increasing incubation time. Moreover, the fluorescence signals from Lyso-Tracker Red and DOX overlapped well after 5 h of incubation. All these results revealed that such drug-loaded vesicles got inside the tumor cells by endocytosis and co-localized in lysosomes, where acidic tumor environment caused the disaggregation of such nanocarriers to deliver DOX and G.

Figure 7. CLSM images of MCF-7 cells pre-treated with drug-loaded vesicles for 1 h and 5 h, respectively. Scale bars = 20 µm. Next, the level of drug-loaded vesicles and free DOX·HCl internalized in MCF-7 cancer cells was quantified by flow cytometry. As presented in Figure 8, the intensity of drug-loaded vesicles was obviously higher than DOX·HCl whatever after incubation for 1 h or 5 h with the same DOX concentration, indicating higher cellular uptake activity of drug-loaded vesicles.


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Figure 8. Flow cytometric analysis of fluorescence intensity in MCF-7 cells pre-treated with DOX-loaded vesicles or DOX·HCl for 1 h and 5 h, respectively.

CONCLUSIONS In conclusion, we have synthetized a NIR-absorbing PDI dye G that not only acted as a photothermal agent but also served as a guest. Multifunctional supramolecular vesicles were efficiently fabricated by the recognition of G and WP5, which showed high-loading capacity for chemotherapy drug DOX. The resulting DOX-loaded vesicles had high stability in simulated normal physiological condition but showed rapid DOX release in the acidic tumor microenvironment, enabling them excellent candidate in the fields of controlled drug release. More importantly, in comparison with chemotherapy or PTT treatment alone, cytotoxicity experiments indicated that the synergistic therapy of the supramolecular vesicles achieved a higher therapeutic efficacy against MCF-7 cancer cells. Furthermore, such supramolecular nanocarriers could efficiently get inside the tumor cells by endocytosis to achieve excellent accumulation of drug in tumor sites. This study offers an innovative tactic to fabricate stimuliresponsive DDS, which have promising potential applications in synergistic cancer therapy.

EXPERIMENTAL SECTION


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Materials. Doxorubicin was purchased from Melone Pharmaceutical Co., Ltd., 3,4,9,10Perylenetetracarboxylic dianhydride, 11-Aminoundecanoic acid, and pyrolidine were purchased from Sigma-Aldrich. Synthetic Procedures. Compounds WP5,36 GM,44 145 and 446 were prepared according to literature. Detailed synthetic route for G are shown in Scheme S1. Compound 2: 1 (10.00 g, 18.18 mmol), 11-Aminoundecanoic acid (8.30 g, 41.23 mmol), propanoic acid (45 mL), and N-Methyl pyrrolidone (NMP, 250 mL) were mixed in a round-bottom flask. The above solution was stirred at 85 °C for 4 h. After removal of NMP, crude product compound 2 was collected and directly used for the next reaction without further purification. Compound 3: A solution containing 2 (2.00 g, 2.18 mmol) and pyrolidine (40 mL) was stirred at 55 °C for 12 h. After removal of pyrolidine, crude product 3 was purified to yield a green solid (0.83 g, 42%). 1H NMR (298 K, 400 MHz, CDCl3, ppm) δ: 8.38 (s, 2H), 8.32 (d, 2H), 7.53 (d, 2H), 4.22 (t, 4H), 3.69 (s, 4H), 2.76 (s, 4H), 2.33 (t, 4H), 2.05-1.95 (m, 8H), 1.77-1.74 (m, 4H), 1.63-1.58 (m, 4H), 1.46-1.29 (m, 24H). 13C NMR (298 K, 100 MHz, CDCl3, ppm) δ: 179.6, 164.1, 146.5, 134.1, 129.8, 126.7, 123.8, 122.0, 121.7, 120.8, 119.0, 118.0, 52.2, 40.7, 34.2, 29.52, 29.46, 29.42, 29.3, 29.1, 28.3, 27.2, 25.9, 24.8. MALDI-TOF: m/z calcd. for [M]+ = 896.472, found 896.475. Compound G: 3 (1.35 g, 1.47 mmol), 4 (0.81 g, 4.38 mmol), 4-dimethylaminopyridine (catalytic amount), 1-(3’-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.90 g, 4.68 mmol), and dry dichloromethane/methanol (1:1, v/v, 100 mL) were mixed in a round-bottom flask under argon atmosphere. The mixture was stirred at 55 °C for 12 h. After removal of the solvent, the crude product G was purified to yield a green solid (0.33 g, 18%). 1H NMR (298 K, 400 MHz, CD3OD, ppm) δ: 8.14 (d, 2H), 8.07 (s, 2H), 7.30 (d, 2H), 4.55-4.54 (m, 4H), 4.20-4.17 (m, 4H), 3.73-3.70 (m, 4H), 3.57 (s, 4H), 3.22 (s, 18H), 2.55 (s, 4H), 2.40 (t, 4H), 2.02 (s, 4H), 1.91 (s, 4H), 1.80-1.76 (m, 4H), 1.66-1.62 (m, 4H), 1.47 (s, 8H), 1.36 (s, 16H).

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C NMR (298 K, 100 MHz, CD3OD, ppm) δ: 172.8, 163.6,

163.3, 145.7, 133.1, 128.9, 126.1, 123.3, 121.1, 120.7, 119.9, 118.2, 117.2, 64.7, 57.5, 53.14, 53.11, 53.08, 51.5, 34.0, 33.4, 29.4, 29.3, 29.2, 29.1, 28.9, 28.8, 27.6, 27.0, 25.3, 24.4, 23.0. LR-ESI-MS: m/z calcd. for [M–2Br]2+ = 534.33, found 534.55.


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DOX Loading and Release. A solution containing DOX (0.02 mM, after being desalted by TEA), G (0.1 mM) and WP5 (0.02 mM) was stirred overnight, and was purified by dialysis bags with a molecular weight cutoff of 10000 in distilled water for 3 days to afford DOX-loaded vesicles. DOX-loaded vesicles (8 mL) in deionized water were injected into release medium (2 mL, 0.05 M tris-HCl pH = 7.4, and 0.05 M citrate pH = 6.2 or 4.4) at 37 °C. After specified

interval

time,

the

released

DOX

were

measured

by

fluorescence

spectrophotometry. Photothermal Measurement. The photothermal conversion ability of the supramolecular vesicles was measured using an infrared thermal imaging camera. The supramolecular vesicles with different concentrations (0.025, 0.05, 0.10 and 0.20 mM) were exposed to 730 nm NIR laser (1.5 W/cm2, 600 s). In addition, 0.10 mM supramolecular vesicles were irradiation of 730 nm laser with different power densities (0.5, 1.0 and 1.5 W/cm2). Cell Culture. MCF-7 cancer cells were incubated in the Dulbecco’s modified Eagle’s medium (DMEM) with 50 U/mL-1 streptomycin/penicillin, and 10% fetal bovine serum (FBS) at 37 °C in 5% CO2 humidified atmosphere. Cell Cytotoxicity Assay. The biocompatibilities of as-prepared materials were investigated by MTT experiment. MCF-7 cells (5×103 cells per well) were grown in a 96well plate in complete culture medium at 37 °C. After 24 h incubation, MCF-7 cells were then incubated in fresh DMEM containing different concentration of as-prepared materials (G, blank vesicles and drug-loaded vesicles) and divide into the following groups: (1) without laser irradiation, (2) with laser irradiation (730 nm, 1.5 W/cm2, 7 min). Then these treated cells were further incubated in fresh DMEM for 24h. After that, each well were added with MTT solution and further grown for another 4 h. After removal of supernatant, DMSO was used to dissolve the formazans. The optical absorbance of formazans was measured by microplate reader. The untreated MCF-7 cells were adopted as a control and considered as 100% cellular viability. Cellular Uptake and Intracellular Localization. MCF-7 cells in confocal dishes were first incubated with complete culture medium at 37 °C for 24 h. MCF-7 cells were cultured in medium with DOX-loaded vesicles (1 µg/mL DOX) for 1 h and 5 h,


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respectively. Then, the Lyso-Tracker Red or Hochest was added to label lysosomes or stain nuclei. After wash with fresh medium, all of the cells were investigated using CLSM. Flow Cytometry. MCF-7 cancer cells (5 × 104 cells/well) in dishes were incubated with complete culture medium at 37 °C for 24 h. Then, cells were cultured in medium with drug-loaded vesicles or DOX·HCl for 1 h or 5 h with the concentration of DOX at 1 µg/mL. After trypsinizing and wash with PBS, all of the cells were investigated by flow cytometry.

ASSOCIATED CONTENT Supporting Information. NMR spectra of 3 and G, pH-responsive complexation of WP5⊃GM, job plot and association constant of WP5⊃GM, critical aggregation concentration of G and (WP5)2⊃G, best molar ratio of WP5 and G, ζ-potentials of (WP5)2⊃G, UV-vis-NIR spectra of (WP5)2⊃G and G, photothermal conversion performance of G, the cytotoxicity of DOX against MCF-7 cells (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

Notes There are no conflicts to declare.

ACKNOWLEDGMENT We give thanks to the National Natural Science Foundation of China (Grant Nos. 21602112, 21674048, 21574064 and 21575069), the China Postdoctoral Science Foundation (Grant Nos. 2017M611876 and 2017M621792), the Synergetic Innovation Center for Organic Electronics and Information Displays, and the Natural Science Foundation of Jiangsu Province of China (Grant Nos. BM2012010, BZ2010043, and NY211003), the Scientific Starting Fund from Nanjing University of Posts and Telecommunications (NUPTSF) (Grant No. NY215057) for financial support.


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REFERENCES (1) Lin, H.; Chen, Y.; Shi, J. Nanoparticle-Triggered In Situ Catalytic Chemical Reactions for Tumour-Specific Therapy. Chem. Soc. Rev. 2018, 47, 1938-1958. (2) Yao, X.; Zhu, Q.; Li, C.; Yuan, K.; Che, R.; Zhang, P.; Yang, C.; Lu, W.; Wu, W.; Jiang, X. Carbamoylmannose Enhances the Tumor Targeting Ability of Supramolecular Nanoparticles Formed Through Host-Guest Complexation of A Pair of Homopolymers. J. Mater. Chem. B 2017, 5, 834-848. (3) Yu, G.; Yung, B. C.; Zhou, Z.; Mao, Z.; Chen, X. Artificial Molecular Machines in Nanotheranostics. ACS Nano 2018, 12, 7-12. (4) Zhu, H.; Cheng, P.; Chen, P.; Pu, K. Recent Progress in the Development of Near-Infrared Organic Photothermal and Photodynamic Nanotherapeutics. Biomater. Sci. 2018, 6, 746765. (5) Tang, W.; Yang, Z.; Wang, S.; Wang, Z.; Song, J.; Yu, G.; Fan, W.; Dai, Y.; Wang, J.; Shan, L.; Niu, G.; Fan, Q.; Chen, X. Organic Semiconducting Photoacoustic Nanodroplets for Laser-Activatable Ultrasound Imaging and Combinational Cancer Therapy. ACS Nano 2018, 12, 2610-2622. (6) Feng, L.; Gao, M.; Tao, D.; Chen, Q.; Wang, H.; Dong, Z.; Chen, M.; Liu, Z. CisplatinProdrug-Constructed Liposomes as a Versatile Theranostic Nanoplatform for Bimodal Imaging Guided Combination Cancer Therapy. Adv. Funct. Mater. 2016, 26, 2207-2217. (7) Jung, H. S.; Verwilst, P.; Sharma, A.; Shin, J.; Sessler, J. L.; Kim, J. S. Organic MoleculeBased Photothermal Agents: an Expanding Photothermal Therapy Universe. Chem. Soc. Rev. 2018, 47, 2280-2297. (8) Qi, J.; Fang, Y.; Kwok, R. T. K.; Zhang, X.; Hu, X.; Lam, J. W. Y.; Ding, D.; Tang, B. Z. Highly Stable Organic Small Molecular Nanoparticles as an Advanced and Biocompatible Phototheranostic Agent of Tumor in Living Mice. ACS Nano 2017, 11, 7177-7188. (9) Lyu, Y.; Zeng, J.; Jiang, Y.; Zhen, X.; Wang, T.; Qiu, S.; Lou, X.; Gao, M.; Pu, K. Enhancing Both Biodegradability and Efficacy of Semiconducting Polymer Nanoparticles for Photoacoustic Imaging and Photothermal Therapy. ACS Nano 2018, 12, 1801-1810. (10) Guo, B.; Sheng, Z.; Hu, D.; Li, A.; Xu, S.; Manghnani, P. N.; Liu, C.; Guo, L.; Zheng, H.; Liu, B. Molecular Engineering of Conjugated Polymers for Biocompatible Organic


16

Page 17 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nanoparticles with Highly Efficient Photoacoustic and Photothermal Performance in Cancer Theranostics. ACS Nano 2017, 11, 10124-10134. (11) Jiang, Y.; Cui, D.; Fang, Y.; Zhen, X.; Upputuri, P. K.; Pramanik, M.; Ding, D.; Pu, K. Amphiphilic Semiconducting Polymer as Multifunctional Nanocarrier for Fluorescence/Photoacoustic Imaging Guided Chemo-Photothermal Therapy. Biomaterials 2017, 145, 168-177. (12) Miao, Q.; Xie, C.; Zhen, X.; Lyu, Y.; Duan, H.; Liu, X.; Jokerst, J. V.; Pu, K. Molecular Afterglow Imaging with Bright, Biodegradable Polymer Nanoparticles. Nat. Biotechnol. 2017, 35, 1102-1110. (13) Fan, Q.; Cheng, K.; Yang, Z.; Zhang, R.; Yang, M.; Hu, X.; Ma, X.; Bu, L.; Lu, X.; Xiong, X.; Huang, W.; Zhao, H.; Cheng, Z. Perylene-Diimide-Based Nanoparticles as Highly Efficient Photoacoustic Agents for Deep Brain Tumor Imaging in Living Mice. Adv. Mater. 2015, 27, 843-847. (14) Hu, X.; Lu, F.; Chen, L.; Tang, Y.; Hu, W.; Lu, X.; Ji, Y.; Yang, Z.; Zhang, W.; Yin, C.; Huang, W.; Fan, Q. Perylene Diimide-Grafted Polymeric Nanoparticles Chelated with Gd3+ for Photoacoustic/T1-Weighted Magnetic Resonance Imaging-Guided Photothermal Therapy. ACS Appl. Mater. Interfaces 2017, 9, 30458-30469. (15) Yu, G.; Yang, Z.; Fu, X.; Yung, B. C.; Yang, J.; Mao, Z.; Shao, L.; Hua, B.; Liu, Y.; Zhang, F.; Fan, Q.; Wang, S.; Jacobson, O.; Jin, A.; Gao, C.; Tang, X.; Huang, F.; Chen, X. Polyrotaxane-Based Supramolecular Theranostics. Nat. Commun. 2018, 9, 766. (16) Zhang, Y.; Qu, Q.; Cao, X.; Zhao, Y. NIR-Absorbing Dye Functionalized Hollow Mesoporous Silica Nanoparticles for Combined Photothermal-Chemotherapy. Chem. Commun. 2017, 53, 12032-12035. (17) Luo, D.; Carter, K. A.; Geng, J.; He, X.; Lovell, J. F. Short Drug-Light Intervals Improve Liposomal Chemophototherapy in Mice Bearing MIA PaCa-2 Xenografts. Mol. Pharmaceutics 2018, DOI: 10.1021/acs.molpharmaceut.8b00052. (18) Zhang, M.; Zhang, L.; Chen, Y.; Li, L.; Su, Z.; Wang, C. Precise Synthesis of Unique Polydopamine/Mesoporous Calcium Phosphate Hollow Janus Nanoparticles for ImagingGuided Chemo-Photothermal Synergistic Therapy. Chem. Sci. 2017, 8, 8067-8077.


17


Page 18 of 21

(19) Chen, G.; Ma, B.; Wang, Y.; Xie, R.; Li, C.; Dou, K.; Gong, S. CuS-Based Theranostic Micelles for NIR-Controlled Combination Chemotherapy and Photothermal Therapy and Photoacoustic Imaging. ACS Appl. Mater. Interfaces 2017, 9, 41700-41711. (20) Xu, H.; Wang, T.; Yang, C.; Li, X.; Liu, G.; Yang, Z.; Singh, P. K.; Krishnan, S.; Ding, D. Supramolecular Nanofibers of Curcumin for Highly Amplified Radiosensitization of Colorectal Cancers to Ionizing Radiation. Adv. Funct. Mater. 2018, 28, 1707140. (21) Chen, C.; Song, Z.; Zheng, X.; He, Z.; Liu, B.; Huang, X.; Kong, D.; Ding, D.; Tang, B. Z. AIEgen-Based Theranostic System: Targeted Imaging of Cancer Cells and Adjuvant Amplification of Antitumor Efficacy of Paclitaxel. Chem. Sci. 2017, 8, 2191-2198. (22) Chi, X.; Zhang, H.; Vargas-Zúñiga, G. I.; Peters, G. M.; Sessler, J. L. A Dual-Responsive Bola-Type Supra-amphiphile Constructed from a Water-Soluble Calix[4]pyrrole and a Tetraphenylethene-Containing Pyridine Bis-N-oxide. J. Am. Chem. Soc. 2016, 138, 58295832. (23) Wang, K.; Guo, D.-S.; Zhao, M.-Y.; Liu, Y. A Supramolecular Vesicle Based on the Complexation of p-Sulfonatocalixarene with Protamine and its Trypsin-Triggered Controllable-Release Properties. Chem. Eur. J. 2016, 22, 1475-1483. (24) Park, K. M.; Baek, K.; Ko, Y. H.; Shrinidhi, A.; Murray, J.; Jang, W. H.; Kim, K. H.; Lee, J. S.; Yoo, J.; Kim, S.; Kim, K. Mono-allyloxylated Cucurbit[7]uril Acts as an Unconventional Amphiphile To Form Light-Responsive Vesicles. Angew. Chem., Int. Ed. 2018, 57, 3132-3136. (25) Xing, P.; Zhao, Y. Supramolecular Vesicles for Stimulus-Responsive Drug Delivery. Small Methods 2018, 1700364. (26) Murray, J.; Kim, K.; Ogoshi, T.; Yao, W.; Gibb, B. C. The Aqueous Supramolecular Chemistry of Cucurbit[n]urils, Pillar[n]arenes and Deep-Cavity Cavitands. Chem. Soc. Rev. 2017, 46, 2479-2496. (27) Wang, Q.; Cheng, M.; Tian, L.; Fan, Q.; Jiang, J. Supramolecular Polymers Based on a Pillar[5]arene-fused Cryptand: Design, Fabrication and Degradation Accompanied by a Fluorescence Change. Polym. Chem. 2017, 8, 6058-6063. (28) Xiao, T.; Wang, L. Recent Advances of Functional Gels Controlled by Pillar[n]arene-Based Host-Guest Interactions. Tetrahedron Lett. 2018, 59, 1172-1182.


18

Page 19 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(29) Yang, X.; Cai, W.; Dong, S.; Zhang, K.; Zhang, J.; Huang, F.; Huang, F.; Cao, Y. Fluorescent Supramolecular Polymers Based on Pillar[5]arene for OLED Device Fabrication. ACS Macro Lett. 2017, 6, 647-651. (30) Yuan, B.; Xu, J.-F.; Sun, C.-L.; Nicolas, H.; Schönhoff, M.; Yang, Q.-Z.; Zhang, X. Pillar[6]arene Containing Multilayer Films: Reversible Uptake and Release of Guest Molecules with Methyl Viologen Moieties. ACS Appl. Mat. Interfaces 2016, 8, 3679-3685. (31) Xiao, T.; Zhong, W.; Zhou, L.; Xu, L.; Sun, X.-Q.; Elmes, R. B. P.; Hu, X.-Y.; Wang, L. Artificial light-harvesting systems fabricated by supramolecular host-guest interactions. Chin. Chem. Lett. 2018, DOI: 10.1016/j.cclet.2018.05.034. (32) Chen, J.-F.; Lin, Q.; Yao, H.; Zhang, Y.-M.; Wei, T.-B. Pillar[5]arene-Based Multifunctional Supramolecular Hydrogel: Multistimuli Responsiveness, Self-Healing, Fluorescence Sensing, and Conductivity. Mater. Chem. Front. 2018, 2, 999-1003. (33) Zhou, J.; Yu, G.; Huang, F. Supramolecular Chemotherapy Based on Host-Guest Molecular Recognition: a Novel Strategy In The Battle Against Cancer with A Bright Future. Chem. Soc. Rev. 2017, 46, 7021-7053. (34) Meng, L.-B.; Zhang, W.; Li, D.; Li, Y.; Hu, X.-Y.; Wang, L.; Li, G. pH-Responsive Supramolecular Vesicles Assembled by Water-Soluble Pillar[5]arene and a BODIPY Photosensitizer for Chemo-Photodynamic Dual Therapy. Chem. Commun. 2015, 51, 1438114384. (35) Chang, Y.; Yang, K.; Wei, P.; Huang, S.; Pei, Y.; Zhao, W.; Pei, Z. Cationic Vesicles Based on Amphiphilic Pillar[5]arene Capped with Ferrocenium: A Redox-Responsive System for Drug/siRNA Co-Delivery. Angew. Chem., Int. Ed. 2014, 53, 13126-13130. (36) Li, H.; Chen, D.-X.; Sun, Y.-L.; Zheng, Y. B.; Tan, L.-L.; Weiss, P. S.; Yang, Y.-W. Viologen-Mediated Assembly of and Sensing with Carboxylatopillar[5]arene-Modified Gold Nanoparticles. J. Am. Chem. Soc. 2013, 135, 1570-1576. (37) Yang, X.; Wang, D.; Shi, Y.; Zou, J.; Zhao, Q.; Zhang, Q.; Huang, W.; Shao, J.; Xie, X.; Dong, X. Black Phosphorus Nanosheets Immobilizing Ce6 for Imaging-Guided Photothermal/Photodynamic Cancer Therapy. ACS Appl. Mater. Interfaces 2018, 10, 1243112440. (38) Jiang, Y.; Pu, K. Advanced Photoacoustic Imaging Applications of Near-Infrared Absorbing Organic Nanoparticles. Small 2017, 13, 1700710.


19


Page 20 of 21

(39) Zhen, X.; Xie, C.; Jiang, Y.; Ai, X.; Xing, B.; Pu, K. Semiconducting Photothermal Nanoagonist for Remote-Controlled Specific Cancer Therapy. Nano Lett. 2018, 18, 14981505. (40) Li, J.; Xie, C.; Huang, J.; Jiang, Y.; Miao, Q.; Pu, K. Semiconducting Polymer Nanoenzymes with Photothermic Activity for Enhanced Cancer Therapy. Angew. Chem., Int. Ed. 2018, 57, 3995-3998. (41) Zhen, X.; Xie, C.; Pu, K. Temperature-Correlated Afterglow of a Semiconducting Polymer Nanococktail for Imaging-Guided Photothermal Therapy. Angew. Chem., Int. Ed. 2018, 57, 3938-3942. (42) Liu, T.; Zhang, M.; Liu, W.; Zeng, X.; Song, X.; Yang, X.; Zhang, X.; Feng, J. Metal Ion/Tannic Acid Assembly as a Versatile Photothermal Platform in Engineering Multimodal Nanotheranostics for Advanced Applications. ACS Nano 2018, 12, 3917-3927. (43) Guo, S.; Liang, T.; Song, Y.; Cheng, M.; Hu, X.-Y.; Zhu, J.-J.; Wang, L. Supramolecular Polymersomes Constructed from Water-Soluble Pillar[5]arene and Cationic Poly(glutamamide)s and Their Applications in Targeted Anticancer Drug Delivery. Polym. Chem. 2017, 8, 5718-5725. (44) Yu, G.; Yang, J.; Xia, D.; Yao, Y. An Enzyme-Responsive Supra-Amphiphile Constructed by Pillar[5]arene/Acetylcholine Molecular Recognition. RSC Adv. 2014, 4, 18763-18771. (45) Würthner, F.; Stepanenko, V.; Chen, Z.; Saha-Möller, C. R.; Kocher, N.; Stalke, D. Preparation and Characterization of Regioisomerically Pure 1,7-Disubstituted Perylene Bisimide Dyes. J. Org. Chem. 2004, 69, 7933-7939. (46) Kawai, K.; Kaneko, K.; Yonezawa, T. Hydrophilic Quaternary Ammonium Type Ionic Liquids. Systematic Study of the Relationship among Molecular Structures, Osmotic Pressures, and Water-Solubility. Langmuir 2011, 27, 7353-7356.


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